This is a revised application for a project focused on development of an effective vaccine for prevention of disease caused by Ebola virus (EBOV), a filovirus which is an etiologic agent of highly lethal hemorrhagic fever. Ebola virus can be transmitted via person-to-person contact, thus posing a high threat of causing an epidemic outbreak. Our group has been engaged in the development of VLP vaccines against virus infection, and our results have shown that EBOV VLPs produced in insect cells using the recombinant baculovirus expression system, which gives high VLP production yield, exhibit DC-stimulating activity and induce strong antibody responses that neutralize EBOV GP mediated virus infection (Ye et al., 2006), indicating that such VLPs could serve as safe and effective vaccines to induce protective immunity against EBOV infection. Furthermore, we also made the finding in recent studies that immunization with a mixture of DNA and VLP vaccines (DNA/VLP) induced higher levels of both antibody and cellular immune responses in comparison to immunization with DNA or VLP vaccines alone. The combined benefit of two non-viral-vector based vaccine platforms obtained with this novel vaccine strategy demonstrates its potential for the development of an efficacious vaccine against EBOV infection. In this project, we will test the hypotheses that the novel DNA/VLP vaccine strategy will elicit strong cellular and antibody responses against EBOV infection and that the use of an adjuvant will further augment induction of such responses, with the aim to obtain a vaccine strategy that can confer rapid as well as long lasting protection against EBOV infection. Specific Aim 1. We will compare immune responses induced by DNA/VLP immunization with DNA or VLP vaccines alone and determine whether the DNA/VLP vaccine strategy is superior to confer rapid (with reduced number of vaccinations) as well as long lasting (at six months after vaccinations) protection against EBOV infection. Moreover, we will employ a transgenic mouse model to investigate the underlying mechanism for the induction of enhanced antibody and T cell responses by DNA/VLP immunization, which will provide instructive information for further development of more potent vaccines against EBOV as well as other pathogens. Specific Aim 2. We will investigate the effect of adjuvant on DNA/VLP vaccination in comparison with DNA or VLP vaccination alone, and determine whether a potent vaccine strategy can be obtained to elicit protective immunity against EBOV infection by a single immunization. Moreover, we will further investigate the efficacy of DNA/VLP vaccines in guinea pigs to assess their potency against EBOV infection in a different small laboratory animal model, with the aim to determine the potential of the DNA/VLP vaccine strategy for future evaluation in the non-human primate model. Ebola virus (EBOV) is a filovirus which is an etiologic agent of highly lethal hemorrhagic fever. Ebola virus can be transmitted via person-to-person contact, thus posing a high threat of an epidemic outbreak. We have previously shown the combined benefit of two non-viral-vector based vaccine platforms and its potential for the development of an efficacious vaccine against EBOV infection. In this project we will test the hypotheses that the novel DNA/VLP vaccine strategy will elicit strong cellular and antibody responses against EBOV infection and that the use of an adjuvant will further augment induction of such responses.